Exodisc for Rapid, Size-Selective, and Efficient Isolation and Analysis of Nanoscale Extracellular Vesicles from Biological Samples.

Extracellular vesicles (EVs) are cell-derived, nanoscale vesicles that carry nucleic acids and proteins from their cells of origin and show great potential as biomarkers for many diseases, including cancer. Efficient isolation and detection methods are prerequisites for exploiting their use in clinical settings and understanding their physiological functions. Here, we presented a rapid, label-free, and highly sensitive method for EV isolation and quantification using a lab-on-a-disc integrated with two nanofilters (Exodisc). Starting from raw biological samples, such as cell-culture supernatant (CCS) or cancer-patient urine, fully automated enrichment of EVs in the size range of 20-600 nm was achieved within 30 min using a tabletop-sized centrifugal microfluidic system. Quantitative tests using nanoparticle-tracking analysis confirmed that the Exodisc enabled >95% recovery of EVs from CCS. Additionally, analysis of mRNA retrieved from EVs revealed that the Exodisc provided >100-fold higher concentration of mRNA as compared with the gold-standard ultracentrifugation method. Furthermore, on-disc enzyme-linked immunosorbent assay using urinary EVs isolated from bladder cancer patients showed high levels of CD9 and CD81 expression, suggesting that this method may be potentially useful in clinical settings to test urinary EV-based biomarkers for cancer diagnostics.

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

An electrochemical-sensor system for real-time flow measurements in porous materials.

Flow monitoring in porous materials is critical for the engineering of paper-based microfluidic bioassays. Here, we present an electrochemical-sensor system that monitors the liquid flow in porous materials without affecting the real flow in paper-strip samples. The developed microfluidic sensor records an amperometric signal created by the solution movement mediated by paper wicking. This approach allows the in situ monitoring of the different hydrodynamic conditions of a specific paper geometry or composition. In addition, the method proposed in this work was employed to characterise the fluid flow of different nitrocellulose paper strips after oxygen-plasma treatment or dextran coating. The dextran fluid-flow modifiers were further used on the paper strip-based assays as means of signal enhancement. The proposed electrochemical-sensing method offers a valuable alternative to existing optical-based monitoring techniques for flow measurement in paper-based microfluidic systems.

Electrospun TiO2 nanofiber integrated lab-on-a-disc for ultrasensitive protein detection from whole blood.

ELISA-based devices are promising tools for the detection of low abundant proteins in biological samples. Reductions of the sample volume and assay time as well as full automation are required for their potential use in point-of-care diagnostic applications. Here, we present a highly efficient lab-on-a-disc composed of a TiO2 nanofibrous mat for sensitive detection of serum proteins with a broad dynamic range, with only 10 µL of whole blood within 30 min. The TiO2 nanofibers provide high specific surface area as well as active functional groups to capture large amounts of antibodies on the surface. In addition, the device offers efficient mixing and washing for improving the signal-to-noise ratio, thus enhancing the overall detection sensitivity. We employ the device for the detection of cardiac biomarkers, C-reactive protein (CRP) and cardiac troponin I (cTnI), spiked in phosphate-buffered saline (PBS) as well as in serum or whole blood. The device exhibited a wide dynamic range of six orders of magnitude from 1 pg mL(-1) (~8 fM) to 100 ng mL(-1) (~0.8 pM) and a low detection limit of 0.8 pg mL(-1) (~6 fM) for CRP spiked in CRP-free serum and a dynamic range of 10 pg mL(-1) (~0.4 pM) to 100 ng mL(-1) (~4 nM) with a detection limit of 37 pg mL(-1) (~1.5 pM) for cTnI spiked in whole blood.